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  • S1

    Electronic Supplementary Information

    Programmed stereoselective assembly of DNA-binding helical metallopeptides Ilaria Gamba, Gustavo Rama, Elizabeth Ortega-Carrasco, Jean-Didier Maréchal, José Martínez-Costas, M. Eugenio Vázquez* and Miguel Vázquez López*

    I. Gamba, G. Rama, Prof. M. Vázquez López. Departamento de Química Inorgánica and Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS). Universidade de Santiago de Compostela, 15782 Santiago de Compostela. Spain. E-mail: miguel.vazquez.lopez@usc.es

    E. Ortega-Carrasco, Dr. J.-D. Maréchal. Departament de Química. Universitat Autònoma de Barcelona, 08193 Cerdanyola. Spain.

    Prof. J. Martínez-Costas. Departamento de Bioquímica y Biología Molecular, and Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS). Universidade de Santiago de Compostela, 15782 Santiago de Compostela. Spain.

    Prof. M. E. Vázquez. Departamento de Química Orgánica and Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS). Universidade de Santiago de Compostela, 15782 Santiago de Compostela. Spain. E-mail: eugenio.vazquez@usc.es

    Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2014

  • S2

    1. General All reagents were acquired from commercial sources: Dimethylformamide (DMF) and Trifluoroacetic acid (TFA) were purchased from Scharlau, CH2Cl2 from Panreac, CH3CN from Merck, HBTU (O-Benzotriazole- N,N,N',N'-tetramethyl-uronium-hexafluorophosphate) and HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate methanaminium) from GL Biochem (Shanghai) Ltd. All other chemicals were purchased from Sigma-Aldrich or Fluka. All solvents were dry and synthesis grade, unless specifically noted. (NH4)Fe2(SO4)2.6H2O salt from Sigma-Aldrich was used as Fe(II) ion source.

    Reactions were followed by analytical RP-HPLC with an Agilent 1100 series LC/MS using a Luna C18 (250 x 4.60 mm) analytical column from Phenomenex. Standard conditions for analytical RP-HPLC consisted on a linear gradient from 15% to 95% of solvent B for 30 min at a flow rate of 1 mL/min (A: water with 0.1% TFA, B: acetonitrile with 0.1% TFA). Compounds were detected by UV absorption at 222, 254 and 310 nm. High-Performance Liquid Chromatography (HPLC) was performed using an Agilent 1100 series Liquid Chromatograph Mass Spectrometer system. Analytical HPLC was run using a Luna C18 (250 x 4.60 mm) reverse phase analytical column; compounds were detected by UV absorption at 222, 254 and 310 nm. The purification of the peptides was performed on a Luna C18 (250 x 10 mm) semi-preparative reverse phase column from Phenomenex. The standard gradient used for analytical and semi-preparative HPLC was 90:10 to 50:50 over 30 min (water/acetonitrile, 0.1% TFA). Electrospray Ionization Mass Spectrometry (ESI/MS) was performed with an Agilent 1100 Series LC/MSD VL model in positive scan mode using direct injection of the purified peptide solution into the MS. Peptides purification was performed by semi-preparative RP- HPLC with an Agilent 1100 series Liquid Chromatograph using a Luna 5u C18(2) 100A (5 µm, 10 × 250 mm) reverse-phase column from Phenomenex. Standard conditions for analytical and semi-preparative RP- HPLC consisted on an isocratic regime during the first 5 min, followed by a linear gradient from 10% to 50% of solvent B for 30 min (A: water 0.1% TFA, B: acetonitrile 0.1% TFA). Compounds were detected by UV absorption (222 nm) and by ESI+−MS. The fractions containing the products were freeze-dried, and their identity was confirmed by ESI+−MS and MALDI-TOF.

    Electrospray Ionization Mass Spectrometry (ESI/MS) was performed with an Agilent 1100 Series LC/MS model in positive scan mode using direct injection of the purified peptide solution into the MS.

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was performed with a Bruker Autoflex MALDI/TOF model in positive scan mode by direct irradiation of the matrix-absorbed peptide.

    UV measurements were made in a Jasco V-630 spectrophotometer coupled to a Jasco ETC-717 temperature controller, using a standard Hellma semi-micro cuvette (140.002-QS). Measurements were made at 20 ºC.

    Luminescence experiments were made with a Jobin-Yvon Fluoromax-3 fluorescence spectrometers (DataMax 2.20), coupled to a Wavelength Electronics LFI−3751 temperature controller. All measurements were made with a Hellma semi-micro cuvette (114F-QS) at 20 ºC.

    Circular dichroism measurements were made with a Jasco J-715 coupled to a Neslab RTE-111 termostated water bath, using a Hellma 100-QS cuvette (2 mm light pass). Measurements were made at 20 ºC. Samples contained (300 µL) 10 mM PBS buffer (pH = 7.2), 50 μM of peptides; the successive additions of Fe(II) aliquots ((NH4)2Fe(SO4)2·6H2O, ammonium iron(II) sulfate (Mohr's salt), 3 µL, 10 mM aqueous solution) complete the complex formation (Fe(II), 100 µM). The spectra are the average of 2 scans and were processed using the program Sigmaplot 11.0 (Systat Software Inc).

  • S3

    2. Synthesis of the unnatural coordinating residue Fmoc-O1PenBpy-OH (1) The bipyridine residue was obtained following the route previously reported by us,1 and which is based on a method reported by Newkome et al.2, starting from 5,5'-diethyl-2,2'-bipyridine, which was obtained from 5,5-dimethil-2,2-bipyridine using the method reported by Francis H. Case3 and Whittle et al.4

    Scheme S1. Synthesis of the coordinating amino acid 1.

    N N H N

    CO2H

    N N Me Me

    1. KMnO4 / H2O 2. EtOH / H2SO4 3. H2N-NH2 / EtOH toluene, 80 ºC

    N N CO2Et

    1. HCl / NaNO2 2. reflux, EtOH/Xylene

    N N N H

    CO2EtEtO

    O EtOH / NaOH

    a) SOCl2

    b) 2, DIEA CH2Cl2

    N N H2N CO2H

    2

    NH

    O

    H2N

    FmocHN O O

    OH O

    O

    NHFmoc

    Fmoc-O1PenBpy-OH (1)Fmoc-O1Pen-OH

  • S4

    3. Peptide synthesis All peptide synthesis reagents and Fmoc amino acid derivatives were purchased from GL Biochem (Shanghai) Ltd. Novabiochem. Fmoc-O1Pen-OH was purchased from IRIS Biotech (Cat. #: FAA1565).

    C-terminal amide peptides were synthesized following standard peptide synthesis protocols (Fmoc/tBu strategy) on a 0.05 mmol scale using a 0.48 mmol/g loading Fmoc-PAL-PEG-PS resin from Life technologies with a PS-3 automatic peptide synthesizer from Protein Tecnologies. The amino acids were coupled in 5-fold excess using HBTU as activating agent, except for the synthetic Fmoc-O1PenBpy-OH coordinating residue, which was coupled using HATU as activating agent.

    Each amino acid was activated for 30 seconds in DMF before being added onto the resin and couplings were conducted for 30 min. Deprotection of the temporal Fmoc protecting group was performed by treating the resin with 20% piperidine in DMF for 10 min.

    General procedure for cleavage-deprotection: A resin-bound peptide dried overnight (0.025 mmol) was placed in a 50 mL falcon tube, to which 3 mL of the cleavage cocktail (50 µL CH2Cl2, 25 µL of H2O, 25 µL of TIS (Triisopropylsilane) and 940 TFA µL) were added. The resulting suspension was shaken for 3 h. The resin was filtered, and the TFA filtrate was concentrated with a nitrogen current to an approximate volume of 1 mL, which was added to ice-cold diethyl ether (10 mL). After 10 min, the precipitate was centrifuged and washed again with 15 mL of ice-cold ether. The solid residue was dried under argon and redissolved in acetonitrile/water 1:1 (1 mL) and purified by semi-preparative reverse-phase HPLC. The collected fractions were lyophilized and stored at −20 °C. Their identity was confirmed by ESI+-MS and MALDI-TOF.

    LL-H:

    ESI-MS (m/z) [M+H]+ calc for C108H113N31O24 = 2227.8; found = 1114.6, calc. for [M+2H]2+ = 1114.9; found = 743.2, calc. for [M+3H]3+ = 746.6.

    UV (H20) λmax, nm (ε): 308 (169700) M−1cm−1

    Yield: 52%

    DD-H:

    ESI-MS (m/z) [M+H]+ calc for C108H113N31O24 = 2227.8; found = 1114.6, calc. for [M+2H]2+ = 1114.9; found = 743.2, calc. for [M+3H]3+ = 746.6.

    UV (H20) λmax, nm (ε): 308 (169700) M−1cm−1

    Yield: 43%

    Synthesis of the fluorescent peptides

    The coupling of the peptide with the rhodamine fluorescent probe was performed directly on the solid support: 5-(and-6)-carboxy-X-rhodamine (6.7 mg, 9.6 µmol), activated as succinimidyl ester, was dissolved

  • S5

    in 1.5 mL of DIEA solution in DMF (0.19 M, 0.29 mmol). The mixture was sonicated during 2 minutes and then loaded in a SPPS reactor, containing the Rink Amide resin (40 mg, 9.5 µmol) where the LL-H or DD-H peptides were previously grown, as described before. The mixture was gently shaken during 2 hours at rt. The resin was extensively washed and dried. The desired peptide was cleaved from the solid support, using the standard acidic cocktail (5 mL, 2.5 hrs), and purified by HPLC.

    LL-RhH:

    ESI-MS (m/z) [M+H]+ calc for C141H142N33O28 = 2745,07; found = 1374.1, calc. for [M+2H]2+ = 1373,5; found = 915.8, calc. for [M+3H]3+ = 916.0. UV (H20) λmax, nm (ε): 575 (81000) M−1cm−1.

    Yield: 54%

    DD-RhH:

    ESI-MS (m/z) [M+H]+ calc for C141H142N33O28 = 2745,07; found = 1374.1, calc. for [M+2H]2+ = 1373,5; found = 915.8, calc. for [M+3H]3+ = 916.0. UV (H20) λmax, nm (ε): 575 (81,000) M−1cm−1.

    Yield: 48%

    The above described protocol was also followed to synthetize t